Novel Aureobasidium sp. microorganisms and method for obtaining the same, and method for preparing erythritol with the same

ABSTRACT

Biologically pure culture of Aureobasidium sp. SN-124A strain (FERM BP-1429) and artificial mutants thereof according to the present invention show the properties of forming and accumulating erythritol in a culture solution, when aerobically cultured on a liquid culture medium containing an assimilable carbon source and an assimilable nitrogen source, and are useful for the preparation of erythritol by fermentation of sugars. A method for preparing erythritol by fermentation of sugars according to the present invention comprises inoculating microorganism selected from the group consisting of Aureobasidium sp. SN-124A strain (FERM BP-1429) and mutants thereof on a liquid culture medium of pH 4 to 9 containing an assimilable carbon source and an assimilable nitrogen source, and aerobically culturing them at a temperature of 30° to 38° C. to form and accumulate erythritol in said culture medium for collection.

This is a division of application Ser. No. 07/088,858, filed Aug. 24,1987, now U.S. Pat. No. 4,939,091, issued July 3, 1990.

BACKGROUND OF THE INVENTION

The present invention relates to novel microorganisms, a method forobtaining the same, and a method for preparing erythritol byfermentation, using the same. More specifically, the present inventionrelates to Aureobasidium sp. SN-124A strain and its artificial mutants,Aureobasidium sp. SN-G42 and SN-γ96, a method for obtaining suchmicroorganisms, and a method for preparing erythritol by convertingfermentable sugars to erythritol with said microorganisms.

It is well-known to prepare erythritol by fermentation, usingmicroorganisms. The microorganisms so far known to be capable ofproducing erythritol include, for instance, those belonging toDebaryomyces sp. (U.S. Pat. No. 2,986.495), Pichia sp. (U. S. Pat. No.2,986,495), Candida sp. (U. S. Pat. No. 3,756,917), Moniliella sp.(Antonie van Leeuwenhoek, 37(1971)107-118 and such), Aureobasidium sp.(Japanese Patent Laid-Open Publication No. 61-31091) and the like.

However, such known microorganisms have not still been used on anindustrial scale due to their grave disadvantage that cannot practicallybe neglected.

More exactly, U.S. Pat. No. 2,986,495 discloses a method for preparingarabitol, glycerol and erythritol from monosaccharides with the use ofPichia sp. and Debaryomyces sp. microorganisms. According to thismethod, however, only a small amount of erythritol may be produced asthe byproduct, which should be separated out and collected withconsiderable difficulty.

U. S. Pat. No. 3,756,917 teaches a method for the preparation oferythritol from hydrocarbons using Canadian sp. microorganisms. However,this method is of low productivity and so uneconomical due to the needthat the substrate concentration be at most 20 %. Another disadvantageis that the product cannot be used for foods, since there is alikelihood that the starting hydrocarbons may remain in the product.

Antonie van Leeuwenhoek, 37, 107-118 (1971) and Applied Microbiology,12, [3]240-246 (1964) describes a method for preparing erythritol fromglucose with the use of Moniliella (Torula) sp. microorganisms. Thismethod is characterized in that the ratio of conversion of glucose toerythritol is high and the substrate concentration of a culture mediamay be increased to a relatively high level, but has the disadvantagethat a large amount of xanthane gum should be used for defoaming, sincemarked foaming occurs at the time of culture.

Japanese Patent Laid-Open Publication No. 61-31091 describes methods forpreparing erythritol from monosaccharides with the use of Aureobasidiumsp. microorganisms. These methods make it possible to prepare erythritolusing a relatively high substrate concentration of a culture media and,hence, are valuable in its own way.

However, these methods are not always satisfactory owing to thedisadvantages that the yield of erythritol are not only unsatisfactoryrelative to the amount of cells grown by microorganisms, but the optimumpH, temperature and the like are also narrow, and, even if such factorsdepart slightly from the optimum ranges, there is then a remarkable dropof the yield of erythritol.

As a result of screening performed in view of the aforesaidconsiderations and on the erythritol productivity of many microorganismsin order to obtain erythritol-producing microorganisms of highindustrial significance, it has been found that novel microorganismsbelong to Aureobasidium sp. and isolated from a soil within a starchplant in Okinawa Prefecture, Japan, show high erythritol productivity.

SUMMARY OF THE INVENTION

Therefore, a basic object of the present invention is to provide novelmicroorganisms which show high erythritol productivity and is of highindustrial significance.

Another object of the present invention is to provide microorganismswhich can keep high erythritol productivity even in a culture media ofhigh concentration.

A further object of the present invention is to provide microorganismswhich can produce highly hydrophilic cells with no occurrence of foamingduring culture.

A still further object of the present invention is to provide methodsfor obtaining such useful microorganisms as mentioned above.

A still further object of the present invention is to provide methodsfor efficiently preparing erythritol with the use of such microorganismsas mentioned above.

Other objects of the present invention will become apparent from thefollowing detailed description.

According to the present invention, the aforesaid objects of the presentinvention are achieved by wild-type Aureobasidium sp. SN-124A strain andSN-G42 and SN-γ96 mutants obtained by artificial mutation of suchSN-124A strain. Further, the aforesaid objects of the present inventionare achieved by inoculating said strains on a culture media containingfermentable sugars, and aerobically culturing them to accumulateerythritol in a culture solution, followed by collection thereof.

DETAILED DESCRIPTION OF THE INVENTION

The inventive microorganism Aureobasidium sp. SN-124A strain waspurified and isolated by the present inventors from the soil within astarch plant in Okinawa Prefecture, Japan, in the conventional manner(Applied Microbiology 12[1]87 (1964), Can. J. Microbiol. 2, 72 (1956)).The vegetative cells take on a unicellular or egg-form shape, and arepropagated by multipolar budding with no formation of any ascospore, andthe mycelia form true hyphae with a number of multi-lateral budding typeconidia occurring at the tip and sides. The strains grow rapidly on a YMagar culture medium so that a white colony turns to a black colonywithin 3 days, form a film in liquid culture, and are capable ofconverting fermentable sugars to the main product erythritol and a smallamount of glycerol.

The inventive microorganisms Aureobasidium sp. SN-G42 and Aureobasidiumsp. SN-γ96 strains were prepared by irradiating and treating saidAureobasidium sp. SN-124A strain (parent strain) with ultraviolet raysand a mutagenic agent, and further irradiating them with gamma rays, ifdesired.

The method for obtaining such mutants will now be explained. SN-124Astrain is aerobically cultured on a liquid culture medium having aglucose concentration of 22 % to obtain a cultured product, which isthen irradiated with ultraviolet rays for a given period of time.Thereafter, the cultured product is applied over and cultured on an agarculture medium having a glucose concentration of 22 % to pick up grownstrains. The thus picked-up strains are aerobically cultured on a liquidculture medium having a glucose concentration of 33.5 % to separate thecells, which are in turn treated with an 1 mg/ml concentration ofN-methyl-N'-nitro-N-nitrosoguanidine in a buffer solution. Subsequently,the thus treated product (cells) is applied over and cultured on an agarculture medium having a glucose concentration of 40 % to pick up grownstrains and thereby obtain Aureobasidium sp. SN-G42 strain.

Next, the thus picked-up strain is aerobically cultured in a liquidculture medium having a glucose concentration of 40 %, and the culturedproduct is irradiated with a given dose of Co gamma rays. Thereafter,the treated cells are applied over and cultured on an agar culturemedium having a glucose concentration of 45 %, and the grown strains arepicked up as Aureobasidium sp. SN-γ96 strain.

Since the thus prepared mutants Aureobasidium sp. SN-G42 and SN-γ96 havea rate of conversion to erythritol higher than that of the parent strain(wild-type) Aureobasidium sp. SN-124A strain, excel in the tolerance ofhigh sugars concentration and produce hydrophilic cells, they arefurther preferred for the industrial production of erythritol.

The Aureobasidium sp. SN-124A strain used in the present invention havethe following mycological properties.

1. State of Growth on Culture Media

1) Microscopic Examination

Size of Vegetative Cells (*1): 4-7×4-15 microns.

Shape of Vegetative Cells (*1) : Hypha- or Yeast-like unicellular oregg-shape.

Propagation of Vegetative Cells (*1) :

Hypha or Multipolar budding of yeast-like cells.

Mycelia (*2) : Mycelia form true hyphae with a number of multi-lateralbudding type conidia occurring at the tip and sides.

Note *1 : Cultured on a YM agar culture medium at 27° C. for 5 days.

*2 Slide culture by potato glucose agar.

Agar Slant (*3)

Growth : Satisfactory.

Gloss : Not observed.

Color Tone: The colony turns from white to black with the lapse of time.

Note *3 : YM agar culture medium.

3) Liquid Culture (*4)

Surface Growth : Formation of thick film.

Degree of Turbidity : Transparence.

Grust : Marked.

Note *4 : YM liquid culture medium

2. Formation of Ascospore

Potato glucose agar culture medium : Not formed.

Corn meal agar culture medium : Not formed.

YM agar culture medium : Not formed.

Carrot extract culture medium : Not formed.

V₈ culture medium : Not formed.

3. Physiological Properties

Oxygen Demand : Aerobic.

Growth Temperature : Up to about 40° C.

Optimum Growth Temperature : 35° to 37° C.

Growth pH : 2.5 to 9.5.

Optimum Growth pH : 4 to 7.

KNO₃ Assimilation (*5) : Found.

(NH₂)SO₄ Assimilation (*5) : Found.

Decomposition of Urea : Found.

Liquefication of Gelatine : Not found.

Formation of Carotenoid : Not found.

Formation of Organic Acids : Found.

Albutin : Not found.

Formation of Starchy Substances : Not found.

Vitamin Demand (*5) : Found.

Glucose Concentration (*6) : 50 % Growth ++. 60 % Growth ++.

Salt Concentration (*7) : 2 % Growth +. 6 % Growth -.

Note *5 : Estimated by the method of J. Lodder et al in which aWickerham's synthetic medium was used.

*6 : Agar Medium

*7 : Liquid Medium

4. Fermentability of Sugars (*5)

    ______________________________________                                        Glucose            ++                                                         Lactose            -                                                          Galactose          -                                                          Melibiose          -                                                          Sucrose            ++                                                         Raffinose          -                                                          Maltose            +                                                          Cellobiose         -                                                          Trehalose          -                                                          Inulin             -                                                          ______________________________________                                    

5. Assimilation of Sugars, Organic Acids and so on

    ______________________________________                                        Glucose            ++                                                         D-Xylose           ±                                                       Galactose          -                                                          Erythritol         +                                                          D-Arabinose        -                                                          L-Arabinose        -                                                          D-Ribose           +                                                          Sucrose            +                                                          L-Rhamnose         -                                                          Maltose            +                                                          Ethanol            -                                                          Cellobiose         +                                                          Salicin            -                                                          L-Sorbose          -                                                          Ribitol            -                                                          Galactitol         -                                                          Glycerol           +                                                          Trehalose          -                                                          Lactose            -                                                          Melibiose          -                                                          D-Mannitol         +                                                          Raffinose          -                                                          Melezitose         -                                                          α-Methyl-D-Glucoside                                                                       -                                                          Inulin             ±                                                       Inositol           -                                                          Soluble Starch     -                                                          DL-Lactic Acid     -                                                          Succinic Acid      ±                                                       Citric Acid        +                                                          ______________________________________                                    

As a result of investigations made of the taxonomical position of thepresent strains having such mycological properties as mentioned above,referring to the Genera of Fungi Sporulating in Pure Culture (J. A. VonArx; 1974), it has been found that the present strains are characterizedby forming a colony turning from white to black with the lapse of timeat the later stage of culture on a YM agar medium, forming mycelia onvegetative cells, giving rise to a number of blast spores without anythick sporulation, and so on. Thus, the present strains have been judgedto be novel microorganisms belonging to the genus Aureobasidium andnamed Aureobasidium sp. SN-124A strain.

On the other hand, the mycological properties of Aureobasidium sp.SN-G42 and SN-γ96 strains bear very close resemblance to those of theaforesaid parent Aureobasidium sp. SN-124A strain (wild-type). In otherwords, the mutants have the same mycological properties as those of theparent strain, except that the former gives rise to a colony which iswrinkled on the surface when cultured on an agar culture medium, showslight reduced assimilation of nitrates and are unable to decomposeurea, and that the former is somewhat different from the latter in the"Assimilation of Sugars, Organic Acids and so on" as set forth in Table1.

                  TABLE 1                                                         ______________________________________                                                 Assimilation of Sugars, Oraganic Acids                                        and so on                                                            Substrate  SN-124A     SN-G42     SN-96                                       ______________________________________                                        Glucose    ++          +          +                                           D-Xylose   ±        -          -                                           L-Sorbose  -           +          +                                           D-Mannitol +           -          -                                           Raffinose  -           +          +                                           Melezitose -           +          +                                           Inulin     ±        +          +                                           Succinic Acid                                                                            ±        -          -                                           Citric Acid                                                                              ±        -          -                                           ______________________________________                                    

As can clearly be seen from the foregoing explanation, Aureobasidium sp.SN-G42 and SN-γ96, that are the mutants, have the mycological propertiesbearing very close resemblance to those of Aureobasidium sp. SN-124Astrain that is the parent strain (wild-type). As will be illustrated inthe examples to be given later, however, these mutants are differentfrom the parent, Aureobasidium sp. SN-124A strain in that the highertolerance of sugars concentration, and the cells are so hydrophilic thatfoaming does not substantially occur during culture.

More specifically, Aureobasidium sp. SN-124A strain (wild-type) shows arelatively satisfactory conversion ratio to erythritol of about 37.0 to41.5 % under the condition that the glucose concentration of a culturemedia are up to 33.5 %. However, there is a sharp drop of the convertionratio to erythritol at a glucose concentration exceeding 39.5 %. At aglucose concentration of 45 %, that conversion decreases to about 16 %.With Aureobasidium sp. SN-G42 and SN-γ96 strains, on the other hand, theconversion ratio to erythritol does not virtually drop, even when theglucose concentration of a culture medium exceeds 40 %. At a glucoseconcentration of as high as 45 %, a satisfactory conversion ratio isattained. Particularly with SN-γ96 strain, a high conversion ratiohigher than 40 % is maintained, even when the glucose concentration of aculture medium exceeds 60 %, and a conversion ratio of as high as 32 %is attained, even when the glucose concentration of a culture medium is75 %. Further, SN-γ96 and SN-G42 strains are characterized in that theircells are so hydrophilic that any foaming does not substantially occurduring culture.

The strains according to the present invention have been deposited withFermentation Research Institution of Agency of Industrial Science andTechnology under FERM P-8745 (FERM BP-1429) for Aureobasidium sp.SN-124A strain, FERM P-8940 (FERM BP-1430) for Aureobasidium sp. SN-G42and FERM P-9400 (FERM BP-1431) for Aureobasidium sp. SN-γ96.

In what follows, the methods for preparing erythritol using themicroorganisms according to the present invention will be explained. Inthe specification, it is to be understood that % shall mean w/v %,unless otherwise noted.

The present strains are cultured under aerobic conditions on a liquidculture medium containing an assimilable carbon source, an assimirablenitrogen source, inorganic salts and the like.

For the carbon source of the liquid culture media, use may be made offermentable sugars such as glucose, fructose and sucrose. The upperlimit of the sugar concentration varies depending upon the strains usedand, hence, is not generally determined. However, a sugar concentrationof preferably 10 to 40 %, more preferably 20 to 30 % is used withSN-124A strain. With SN-G42 strain, a sugar concentration of 10 to 55 %,preferably 20 to 50 % is applied, and with SN-γ96 strain, a sugarconcentration of 10 to 95 %, preferably 20 to 70 % is used.

As the nitrogen sources, nitrogen compounds available by miroorganismsare used such as, for instance, yeast extract, malt extract, casaminoacids, corn steep liquor, ammonium sulfate and urea. These nitrogensources are preferably used in an amount of 0.5 to 3.0 % for yeastextract, and 1.5 to 10 % for corn steep liquor.

The inorganic salts used include salts such as ferrous sulfate, sodiumchloride, dipotassium hydrogen phosphate, calcium hydroxide and zincsulfate, and are preferably used in an amount not more than 0.1 %.

It is to be noted that in addition to these carbon sources, nitrogensources and inorganic salts, various organics and inorganics requiredfor the growth of microorganisms or conventionally used defoamers or thelike may be added.

For culture, the cells of the present microorganisms may be inoculateddirectly on the liquid culture media having the aforesaid composition,or a separate seed culture solution obtained by pre-culture may beinoculated thereon. Such a seed culture solution is prepared by theinocuation of a loopfull of yeast-like slant-cultured in theconventional manner on a liquid culture media of pH 4 to 7 containing40.0 % of glucose and 1.6 % of yeast extract, followed by culture at 34°to 37° C. for 2 to 4 days.

Culture is carried out in a temperature range in which microorganismscan grow, i.e., at 30° to 38° C., preferably 35° to 37° C.

The culture media are adjusted to pH 4 to 9, preferably 4 to 7.

The culture period varies depending upon the type of the media used andthe concentration of sugar that provides the carbon source, but isusually about 4 to 15 days. Referring to the mode of culture, it may becarried out either batchwise or continuously.

It is desired that the most use be made of the nutrition source of theculture media, and culture come to an end at the time when the amount oferythritol produced in the culture solution reaches a maximum. For thatpurpose, it is desired that culture be carried out, while measuring theamount of erythritol in the culture solution by means of the knownmethods such as GLC (gas liquid chromatography) and HPLC (highperformance liquid chromatography).

Erythritol accumulated in the culture solution may be separatedtherefrom in the conventional manner after the completion of culture. Tothis end, use may be made of the known means ordinarily employed in theart such as filtration, centrifugation, ion exchange or adsorptionchromatography, solvent extraction, distillation and crystallizationwhich may suitably be combined, if required. By way of example, thecells are removed from the culture solution as by filtration orcentrifugation, and the obtained solution is treated with activatedcharcoal to remove colored matters. Then, that solution is furtherdeionized with an ion exchange resin, and is thereafter concentratedinto a syrup, from which erythritol is finally separated bycrystallization.

The invented novel microorganisms SN-124A strain belonging to the genusAureobasidium and the mutants, Aureobasidium sp. SN-G42 andAureobasidium sp. SN-γ96 show a high erythritol conversion ratio fromsugars and are thus suitable for industrial use. In particular, themutants SN-G42 and SN-γ96 are extremely useful for the industrialpurpose, since they have considerably high tolerance to sugarsconcentration, excel in heat resistance, and give rise to no substantialfoaming, when cultured.

The embodiments of the present invention will now be explained furtherconcretely with reference to the following examples.

EXAMPLE 1 (a) Preparation of Seed Culture Solution

The cells of Aureobasidium sp. SN-124A strain (FERM BP-1429 were appliedover a slant culture medium comprising 20.0 % (w/w) of glucose, 0.5 % ofyeast extract and 1.5 % of agar, and stationary culture was carried outat 35° C. for 3 days.

Then, one platinum loop of the aforesaid cultured cells was transplantedinto a Erlenmeyer flask having a volume of 500 ml loaded therein with100 ml of a liquid culture medium (pH 5.5) containing 20 % (w/w) ofglucose and 2.0 % of corn steep liquor (manufactured by Oji Corn StarchCo., Ltd.), and cultivation was carried out at 35° C. for 3 days toobtain a cultured product.

(b) Main Culture

Five (5) kg of a culture medium containing 20 % (w/w) of glucose, 2.0 %of corn steep liquor preadjusted to pH 6.0, 0.1 % of sorbitan fatty acidester and 0.03 % of a silicon base defoamer were put in a fementerhaving a volume of 7 l, and were then sterilized at 120° C. for 20minutes. After cooled off, pH of the medium was adjusted to 5.5. Addedto this were 200 ml of a seed culture solution of Aureobasidium sp.SN-124A strain (FERM P-8745, FERM BP-1429) prepared in the aforesaidstep (a). That medium was cultured at 35° C. and 400 rpm under anaeration of 1 l/min. for 7 days. As a result, 467 g of erythritol and atrace of glycerol were built up in this culture solution.

The cells were centrifuged from the culture solution, which was in turndecolored with activated charcoal and desalted with an ion exchangeresin (IRA-410: IRA-120B =2 : 1), and was then concentrated and storedat 5° C. to obtain crystals. The crystals were dissolved andrecrystallized in a similar manner. The obtained polyhedral whitecrystals were of a delightful sweet taste and had a melting point of121° C. From the results of NMR, the specific optical rotation, HPLC andGLC, the white crystals were indentified as erythrithol(meso-erythritol).

EXAMPLE 2

Fifty (50) g of a medium containing 30 % (w/w) of glucose and 3.2 % ofcorn steep liquor (adjusted to pH 6.0) were put in a 500 ml Erlenmyerflask, followed by sterilization and cooling. Thereafter, the medium wasadjusted to pH 6.0. Inoculated on this were 2 ml of a seed culturesolution similar to that used in Ex. 1 for 10-day rotary shaking cultureat 35° C. and 180 rpm. As a result, 5.9 g of erythritol and 1.4 g ofglycerol were accumulated in broth.

EXAMPLE 3

Fifty (50) g of a culture medium containing 20 % (w/w) of sucrose and0.5 % of yeast extract were put in a 500 ml Erlenmyer flask, and weresterilized at 120° C. for 15 minutes. After cooling, the medium wasadjusted to pH 6.0, and was inoculated with Aureobasidium sp. SN-124Astrain (FERM BP-1429) for rotary shaking culture at 35° C. and 180 rpmfor 7 days. As a results, 4.2 g of erythritol were accumulated in theculture solution, but any accumulation of glycerol was not found.

EXAMPLE 4

Fifty (50) g of a culture medium containing 0.5 % of yeast extract and10 to 30 % (w/w) of glucose were put in 500 ml Erlenmyer flasks, andwere sterilized at 120° C. for 15 minutes. After cooling, the media wereadjusted to pH 5.5, and were inoculated with 2 ml of a seed culturesolution (obtained by culturing Aureobasidium sp. SN-124A strain (FERMBP-1249) on a culture medium comprising 20 % of glucose and 0.5 % ofyeast extract at 35° C. for 3 days for rotary shaking culture) at 35° C.and 180 rpm for 4 to 11 days. The results were as set forth in Table 1.

                  TABLE 1                                                         ______________________________________                                        Glucose Con-                                                                           Culture                                                              centration                                                                             Periods  Yields* (%)    Glucose                                      (%)      (days)   Erythritol                                                                              Glycerol                                                                             Residues (%)                               ______________________________________                                        10       4        40.6      1.0    0                                          15       6        41.8      2.6    0                                          20       7        46.2      3.4    2.2                                        25       9        42.2      7.8    15.7                                       30       11       37.0      10.6   23.3                                       ______________________________________                                         *per consumed glucose                                                    

EXAMPLE 5

Fifty 50) g of a culture medium comprising 20 % (w/w) of glucose and 0.5% of yeast extract were put in each of eight 500 ml Erlenmyer flasks,and were sterilized. After cooling, the medium was adjusted to pH rangeof 2.9 to 10.1 with 1 N hydrochloric acid or 1 N sodium hydroxide. Eachof the obtained media was then inoculated with seed strains(Aureobasidium sp. SN-124A strain , FERM BP-1429) for 7-day rotaryshaking culture at 35° C. and 180 rpm. As a result, erythritol wasobtained in the yields as set forth in Table 2.

                  TABLE 2                                                         ______________________________________                                        pH of Culture                                                                             Yields* (%)      Glucose                                          Media       Erythritol  Glycerol Residues (%)                                 ______________________________________                                        2.9         35.8        6.4      27.5                                         4.1         42.0        8.1      14.0                                         5.1         46.6        9.2      16.1                                         6.1         45.6        7.2      12.8                                         6.7         46.2        8.0      15.0                                         7.8         47.2        5.0      23.0                                         9.1         41.6        3.0      35.7                                         10.1        1.0         0        81.9                                         ______________________________________                                         *per consumed glucose                                                    

EXAMPLE 6 Preparation of Mutents

Aureobasidium sp. SN-124A strain (FERM BP-1429) was precultured on aculture medium containing 0.5 % of yeast extract and 22 % of glucose for2 days. The pre-cultured product was diluted 100-fold, and wasirradiated with a 15-W ultraviolet lamp (Toshiba Sterilizer Lamp GL15)from a distance of 30 cm for 40 minutes. After dilution, the irradiatedproduct was applied over an agar culture medium (1.0 % of yeast extract,33.5 % of glucose and 1.5 % of agar) to pick up growing strains.

The thus picked-up strain was again irradiated with ultraviolet rays topick up growing strains in a similar manner.

Further, the thus picked-up strain was treated with an 1 mg/mlconcentration of N-methyl-N'-nitro-N"nitrosoguanidine for 30 minutes topick up growing strains in a similar manner and thereby obtainAuroebasidium sp. SN-G42 strain (FERM P-8940, FERM BP-1430) which wasthe mutant.

EXAMPLE 7 (a) Preparation of Seed Culture Solution

The cells of Aureobasidium sp. SN-G42 strain (FERM BP-1430) were appliedover a slant culture medium comprising 33.5 % of glucose, 1.0 % of yeastextract and 1.5 % of agar, and stationary culture was carried out at 35°C. for 2 to 3 days.

Then, a loopfull of the aforesaid cultured cells was transplanted intoan Erlenmeyer flask having a volume of 500 ml loaded therein with 150 mlof a liquid culture medium (pH 4.0) containing 20 % of glucose and 1.6 %of corn steep liquor (manufactured by Oji Corn Starch Co., Ltd.), andcultivation was carried out at 35° C. for 3 days. Further, 9 ml of thisculture solution were inoculated into a 500 ml Erlenmeyer flask loadedtherein with 150 ml of a similar liquid culture medium, and rotaryshaking culture was carried out at 35° C. for 3 days.

(b) Main Culture

Fifteen (15)l of a culture medium containing 20.0 % of glucose and 1.6 %of corn steep liquor were put in a fermeter of 30 l in volume, to which300ppm of a deformer (Silicone KS-66 manufactured by Shinetsu Kagaku,Co., Ltd.) were then added, followed by steam sterilization at 120° C.for 20 minutes. After cooling, the culture medium was adjusted to pH 4.0with caustic soda. Added to this medium were thereafter 6 % of the seedculture solution of Aureobasidium sp. SN-G42 strain (FERM BP-1430).Culture was then carried out at 35° C. and 230 rpm under an aeration of0.25 vvm.

After the completion of culture, the analysis of the culture solutionindicated that the glycerol was entirely consumed, and the yields oferythritol and glycerin therein were 49.3 % and 2.2 %, respectively.

Next, a part of this culture solution was freed of the cells bycentrifugation, and was further decolored and desalted with activatedcharcoal and an ion exchange resin (IRA-410 : IR-120B=2 : 1). Theresulting liquid was concentrated to a sugar concentration of at least50 %, and was slowly cooled to obtain crystals, which were in turndissolved in water and recrystallized in a similar manner.

The thus obtained polyhedral white crystals were of a comfortable sweettaste, and had a melting point of 121° C. From the results of HPLC andGLC and the measurement of the optical rotation and nuclear magneticresonance spectra, the aforesaid crystals were identified as erythritol(meso-erythritol).

EXAMPLE 8

Fifteen (15) l of a culture medium containing 20.0 % of glucose and 1.6% of corn steep liquor were loaded into a fermenter of 30 l in volume,and were added with 300 ppm of a defoamer, followed by steamsterilization at 120° C. for 20 minutes. After cooling, the culturemedium was adjusted to pH 4.0 with caustic soda. Added to this mediumwas thereafter 6 % of a seed culture solution of Aureobasidium sp.SN-G42 strain (FERM BP-1430) prepared with the same medium as theaforesaid medium in accordance with the procedures of Example 7.Cultivation was then carried out at 35° C. and 230 rpm under an aerationof 0.50 vvm for 7 days.

After the completion of cultivation, the analysis of the culturesolution indicated that the glucose was entirely consumed, and theyields of erythritol and glycerol therein were 49.2 % and 1.2 %,respectively.

EXAMPLE 9

Fifteen (15) l of a culture medium containing 20.0 % of glucose and 1.6% of corn steep liquor were loaded into a fermenter of 30 l in volume,and were added with 300 ppm of a defoamer, followed by steamsterilization at 120° C. for 20 minutes. After cooling, pH of theculture medium was adjusted to 4.0 with caustic soda. Added to thismedium was thereafter 6 % of a seed culture solution of Aureobasidiumsp. SN-G42 strain (FERM BP-1430) prepared with the same medium as theaforesaid medium in accordance with the procedures of Example 7. Culturewas then carried out at 35° C. and 230 rpm under an aeration of 0.75 vvmfor 7 days.

After the completion of culture, the analysis of the culture solutionindicated that the glucose was entirely consumed, and the yields oferythritol and glycerol therein were 46.5 % and 2.7 %, respectively.

EXAMPLE 10

Fifteen (15) l of a culture medium containing 20.0 % of glucose and 1.6% of corn steep liquor were loaded into a fermenter of 30 l in volume,and were added with 300 ppm of a defoamer, followed by steamsterilization at 120° C. for 20 minutes. After cooling, the culturemedium was adjusted to pH 4.0 with caustic soda. Added to this mediumwas thereafter 900 ml of a seed culture solution of Aureobasidium sp.SN-G42 strain (FERM BP-1430) prepared with the same medium as theaforesaid medium in accordance with the procedures of Example 7. Culturewas then carried out at 35° C. and 230 rpm under an aeration of 1.00 vvmfor 7 days.

After the completion of culture, the analysis of the culture solutionindicated that the glucose was entirely consumed, and the yields oferythritol and glycerol therein were 41.8 % and 1.4 %, respectively.

EXAMPLE 11

Five (5) l of a culture medium containing 33.5 % of glucose and 4.47 %of corn steep liquor were loaded into a fermenter of 7 l in volume, andwere added with 300 ppm of a defoamer, followed by steam sterilizationat 120° C. for 20 minutes. After cooling, the culture medium wasadjusted to pH 4.2 with caustic soda. Added to this medium wasthereafter 300 ml of a seed culture solution of Aureobasidium sp. SN-G42strain (FERM BP-1430) prepared with the same medium as the aforesaidmedium in accordance with the procedures of Example 7. Culture was thencarried out at 35° C. and 500 rpm under an aeration of 0.25 vvm for 5days.

After the completion of culture, the analysis of the culture solutionindicated that the glucose was entirely consumed, and the yields oferythritol and glycerol therein were 39.7 % and 7.9 %, respectively.

EXAMPLE 12

Five (5) l of a culture medium containing 39.5 % of glucose and 4.0 % ofcorn steep liquor were loaded into a fermenter of 7 l in volume, andwere added with 300 ppm of a defoamer, followed by steam sterilizationat 120° C. for 20 minutes. After cooling, the culture medium wasadjusted to pH 4.2 with caustic soda. Added to this medium wasthereafter 300 ml of a seed culture solution of Aureobasidium sp. SN-G42strain (FERM BP-1430) prepared with the same medium as the aforesaidmedium in accordance with the procedures Example 7. Culture was thencarried out at 35° C. and 500 rpm under an aeration of 0.25 vvm for 6days.

After the completion of culture, the analysis of the culture solutionindicated that glucose was entirely consumed, and the yields oferythritol and glycerol therein were 38.6 % and 9.5 %, respectively.

EXAMPLE 13

Five (5) l of a culture medium containing 39.5 % of glucose and 5.3 % ofcorn steep liquor were loaded into a fermenter of 7 l in volume, andwere added with 300 ppm of a defoamer, followed by steam sterilizationat 120° C. for 20 minutes. After cooling, the culture medium wasadjusted to pH 4.2 with caustic soda. Added to this medium wasthereafter 300 ml of a seed culture solution of Aureobasidium sp. SN-G42strain (FERM BP-1430) prepared with the same medium as the aforesaidmedium in accordance with the procedures of Example 7. Culture was thencarried out at 35° C. and 500 rpm under an aeration of 0.25 vvm for 5days.

After the completion of culture, the analysis of the culture solutionindicated that the glucose was entirely consumed, and the yields oferythritol and glycerol therein were 37.5 % and 7.2 %, respectively.

EXAMPLE 14

Five (5) l of a culture medium containing 39.5 % of glucose and 6.6 % ofcorn steep liquor were loaded into a fermenter of 7 l in volume, andwere added with 300 ppm of a defoamer, followed by steam sterilizationat 120° C. for 20 minutes. After cooling, the culture medium wasadjusted to pH 4.2 with caustic soda. Added to this medium wasthereafter 300 ml of a seed culture solution of Aureobasidium sp. SN-G42strain (FERM BP1430) prepared with the same medium as the aforesaidmedium in accordance with the procedures of Example 7. Culture was thencarried out at 35° C. and 500 rpm under an aeration of 0.25 vvm for 4days.

After the completion of culture, the analysis of the culture solutionindicated that the glucose was entirely consumed, and the yields oferythritol and glycerol therein were 37.1 % and 4.8 %, respectively.

EXAMPLE 15

Fifteen (15) l of a culture medium containing 45.0 % of glucose and 6.0% of corn steep liquor were loaded into a fermenter of 30 l in volume,and were added with 300 ppm of a defoamer, followed by steamsterilization at 120° C. for 20 minutes. After cooling, the culturemedium was adjusted to pH 4.0 with caustic soda. Added to this mediumwas thereafter 6 % of a seed culture solution of Aureobasidium sp.SN-G42 strain (FERM BP-1430) prepared with the same medium as theaforesaid medium in accordance with the procedures of Example 7. Culturewas then carried out at 35° C. and 230 rpm under an aeration of 0.33 vvmfor 8 days.

After the completion of culture, the analysis of the culture solutionindicated that the glucose was entirely consumed, and the yields oferythritol and glycerol therein were 37.3 % and 4.5 %, respectively.

EXAMPLE 16

Five (5) l of a culture medium containing 33.5 % of sucrose and 4.5 % ofcorn steep liquor were loaded into a fermenter of 7 l in volume, andwere added with 300 ppm of a defoamer, followed by steam sterilizationat 120° C. for 20 minutes. After cooling, the culture medium wasadjusted to pH 4.1 with caustic soda. Added to this medium wasthereafter 300 ml of a seed culture solution of Aureobasidium sp. SN-G42strain (FERM BP-1430) prepared with the same medium as the aforesaidmedium in accordance with the procedures of Example 7. Culture was thencarried out at 35° C. and 500 rpm under an aeration of 0.25 vvm for 6days.

After the completion of culture, the analysis of the culture solutionindicated that the sucrose was entirely consumed, and the yields oferythritol and glycerol therein were 41.9 % and 7.4 %, respectively.

EXAMPLE 17

Five (5) l of a culture medium containing 39.5 % of sucrose and 5.3 % ofcorn steep liquor were loaded into a fermenter of 7 l in volume, andwere added with 300 ppm of a defoamer, followed by steam sterilizationat 120° C. for 20 minutes. After cooling, the culture medium wasadjusted to pH 4.1 with caustic soda. Added to this medium wasthereafter 300 ml of a seed culture solution of Aureobasidium sp. SN-G42strain (FERM BP-1430) prepared with the same medium as the aforesaidmedium in accordance with the procedures of Example 7. Culture was thencarried out at 35° C. and 500 rpm under an aeration of 0.25 vvm for 6days.

After the completion of culture, the analysis of the culture solutionindicated that the sucrose was entirely consumed, and the yields oferythritol and glycerol therein were 42.4 % and 5.8 %, respectively.

EXAMPLE 18

Five (5) l of a culture medium containing 45.0 % of sucrose and 6.0 % ofcorn steep liquor were loaded into a fermenter of 7 l in volume, andwere added with 300 ppm of a defoamer, followed by steam sterilizationat 120° C. for 20 minutes. After cooling, the culture medium wasadjusted to pH 4.1 with caustic soda. Added to this medium wasthereafter 300 ml of a seed culture solution of Aureobasidium sp. SN-G42strain (FERM BP-1430) prepared with the same medium as the aforesaidmedium in accordance with the procedures of Example 7. Culture was thencarried out at 35° C. and 500 rpm under an aeration of 0.25 vvm for 6days.

After the completion of culture, the analysis of the culture solutionindicated that the sucrose was entirely consumed, and the yields oferythritol and glycerol therein were 41.7 % and 3.8 %, respectively.

EXAMPLE 19 Preparation of Mutants

Five (5) ml of a liquid culture medium containing 22.0 % of glucose and0.5 % of yeast extract were inoculated with Aureobasidium sp. SN-124Astrain (FERM BP-1429) for 2-day rotary shaking culture at 30° C.,thereby obtaining a cultured product.

Then, the thus cultured product was diluted 100 times with a 22.0 %glucose solution, and was irradiated with an ultraviolet lamp of 15 W(Toshiba Sterilizer lamp GL 15) from a distance of 30 cm for 40 minutes,while stirred in a petri dish.

After the irradiation, the thus treated solution in the petri dish wasapplied over an agar culture medium containing 22.0 % of glucose, 0.5 %of yeast extract and 1.5 % of agar, and stationary culture was carriedout at 30° C. for 4 days to pick up grown strains.

Then, the thus picked-up strains were inoculated on a liquid culturemedium containing 22.0 % of glucose and 0.5 % of yeast extract for 2-dayrotary shaking culture at 30° C., thereby obtaining a cultured product.

Then, this cultured product was diluted in a manner similar to theaforesaid manner, and was thereafter re-irradiated with ultraviolet raysfor 20 minutes under the same conditions as mentioned above.

After the irradiation, the thus treated solution was applied over anagar culture medium containing 33.5 % of glucose, 1.0 % of yeast extractand 1.5 % of agar, and stationary culture was carried out at 30° C. for4 days to pick up grown strains.

Further, the thus picked-up strains were inoculated on a liquid culturemedium containing 33.5 % of glucose and 1.0 % of yeast extract, androtary shaking culture was carried out at 30° C. for 2 days to obtain acultured product.

Next, the cells were centrifuged out of the thus obtained cultureproduct, and were further washed twice with a 0.2 M acetic acid buffersolution (pH 5.0) containing 2 M of glucose. Afterwards, the cells weresuspended in a buffer solution at a concentration of 1×10⁷ cells/ml, andwere treated with N-methyl-N'-nitro-N-nitrosoguanidine having aconcentration of 1 mg/ml at 30° C. for 30 minutes.

After the completion of the treatment, the cells were separated in theconventional manner, and were further washed with a buffer solution.Thereafter, the cells were applied over an agar culture mediumcontaining 40.0 % of glucose, 1.0 % of yeast extract and 1.5 % of agar,and stationary culture was carried out at 30° C. for 5 days to pick upgrown strains.

The thus picked- up strains were inoculated on a liquid culture mediumcomprising 1.6 % of yeast extract and 40.0 % of glucose, and shakingculture was carried out at 30° C. for 2 days to obtain a culturedproduct. Then, the obtained culture product was diluted to 1×10⁷cells/m(, and 3 ml of the diluted product were put in a glass test tube,which was irradiated with 200 kRad of gamma rays (⁶⁰ Co) from a distanceof 18 cm.

After the completion of irradiation, the treated cells were applied overan agar culture medium containing 45.0 % of glucose, 1.6 % of yeastextract and 1.5 % of agar, and stationary culture was carried out at 30°C. for 5 days to pick up grown cells, thereby obtaining mutants, viz.,Aureobasidium sp. SN-γ96 strain (FERM P-9400, FERM BP-1431).

EXAMPLE 20 (a) Preparation of Seed Culture Solution

Aureobasidium sp. SN-γ96 strain (FERM BP-1431) were applied over a slantculture medium comprising 40.0 % of glucose, 1.6 % of yeast extract and1.5 % of agar for 5-day stationary culture at 35° C.

One platinum loop of the aforesaid slant-cultured cells weretransplanted into a 500-ml conical flask, in which 100 ml of a liquidculture medium (pH 4.9) containing 40.0 % of glucose and 1.6 % of yeastextract was loaded, for 3-day culture at 35° C. Further, 9 ml of thisculture solution was inoculated into a 500 ml conical flask in which 150ml of a liquid culture medium similar to the aforesaid one was put,rotary shaking culture was carried out at 35° C. for 3 days to obtain acultured product.

(b) Main Culture

Fifteen (15) l of a liquid culture medium containing 40.0 % of glucoseand 6.8 % of corn steep liquor (which had separately been sterilized)were loaded in a fermenter having a volume of 30 l, added with 300 ppmof a defoamer ("Adecanol LG-109 manufactured by Asahi Denka K.K , andwere adjusted to pH 4.2 with sodium hydroxide. Added to this were 900 mlof the seed culture solution of Aureobasidium sp. SN-γ96 strain (FERMBP-1431) prepared in the aforesaid step (a). Culture was then carriedout at 35° C. and 400 rpm under an aeration of 1.0 vvm for 4 days.

After the completion of culture, the analysis of the culture solution byHPLC indicated that the glucose was completely consumed, and theerythritol and glycerol contents were 18.9 % (the yield : 47.3 %) and3.5 % (the yield : 8.8 %), respectively.

Then, the cells were centrifuged out of 600 ml of this culture solution,which was in turn decolored with activated charcoal and desalted with anion exchange resin (IRA-140 : IR-120=2 : 1). Subsequently, the resultingliquid was concentrated to a sugar concentration of at least 50 %, andwas slowly cooled to precipitate and separate crystals. Further, thecrystals were recrystallized from water to obtain polyhedral whitecrystals which had a melting point of 121.0° C. and were of acomfortable sweet taste. The white crystals were further identified aserythritol (mesoerythritol) by liquid chromatography and gaschromatography as well as the measurement of the optical rotation andnuclear magnetic resonance spectra.

EXAMPLE 21

Fifteen (15) l of a liquid culture medium containing 50.0 % of glucoseand 6.8 % of corn steep liquor (which had separately been sterilized)were loaded in a fermenter having a volume of 30 l, added with 300 ppmof a defoamer, and were adjusted to pH 4.2 with sodium hydroxide. Addedto this were 900 ml of the seed culture solution of Aureobasidium sp.SN-γ96 strain (FERM BP-1431) prepared in accordance with the proceduresof Example 20(a). Culture was then carried out at 35° C. and 400 rpmunder an aeration of 1.5 vvm for 6 days.

After the completion of culture, the analysis of the culture solutionindicated that the glucose was completely consumed, and the erythritoland glycerol contents were 23.3 % (the yield: 46.5 %) and 4.5 % (theyield : 9.0 %), respectively.

EXAMPLE 22

Two (2) l of a liquid culture medium containing 60.2 % of glucose and2.0 % of yeast extract (which had separately been sterilized) wereloaded in a fermenter having a volume of 3 l, and added with 200 ppm ofa defoamer. Added to this were 80 ml of the seed culture solution ofAureobasidium sp. SN-γ96 strain (FERM BP-1431) prepared in accordancewith the procedures of Example 20(a). Culture was then carried out at35° C. and 1000 rpm under an aeration of 2.5 vvm for 7 days.

After the completion of culture, the analysis of the culture solutionindicated that the glucose was completely consumed, and the erythritoland glycerol contents were 28.8 % (the yield : 47.7 %) and 6.8 % (theyield : 11.3 %), respectively.

EXAMPLE 23

Two (2) l of a liquid culture medium containing 75.5 % of glucose and2.0 % of yeast extract (which had separately been sterilized) wereloaded in a fermenter having a volume of 3 l, and added with 200 ppm ofa defoamer. Added to this were 80 ml of the seed culture solution ofAureobasidium sp. SN-γ96 strain (FERM BP-1431) prepared in accordancewith the procedures of Example 20(a). Culture was then carried out at35° C. and 1000 rpm under an aeration of 2.0 vvm for 11 days.

After the completion of culture, the analysis of the culture solutionindicated that the glucose was completely consumed, and the erythritoland clycerol contents were 24.5 % (the yield : 32.4 %) and 11.0 % (theyield : 14.3 %), respectively.

EXAMPLE 24

Five (5) l of a liquid culture medium containing 50.0 % of sucrose and6.8 % of corn steep liquor (which had separately been sterilized) wereloaded in a fermenter having a volume of 7 l, added with 300 ppm of adefoamer, and were adjusted to pH 4.2 with sodium hydroxide. Added tothis were 300 ml of the seed culture solution of Aureobacidium sp.SN-γ96 strain (FERM BP-1431) prepared in accordance with the proceduresof Example 20(a). Culture was then carried out at 35° C. and 800 rpmunder an aeration of 1.0 vvm for 5 days.

After the completion of culture, the analysis of the culture solutionindicated that the sucrose was completely consumed, and the erythritoland glycerol contents were 23.6 % (the conversion ratio : 47.25 %) and5.0 % (the conversion ratio : 10.0 %), respectively.

EXAMPLE 25

Two (2) l of a liquid culture medium containing 61.0 % of sucrose and2.0 % of corn steep liquor (which had separately been sterilized) wereloaded in a fermenter having a volume of 3 l, and added with 200 ppm ofa defoamer. Added to this were 80 ml of the seed culture solution ofAureobasidium sp. SN-γ96 strain (FERM BP-1431) prepared in accordancewith the procedures of Example 20(a). Culture was then carried out at35° C. and 100 rpm under an aeration of 2.5 vvm for 6 days.

After the completion of culture, the analysis of the culture solutionindicated that the sucrose was completely consumed, and the erythritoland glycerol contents were 24.6 % (the yield : 40.3 %) and 7.8 % (theyield : 12.8 %), respectively.

EXAMPLE 26

Two (2) l of a liquid culture medium containing 93.9 % of sucrose and2.0 % of yeast extract (which had separately been sterilized) wereloaded in a fermenter having a volume of 3 l, and added with 200 ppm ofa defoamer. Added to this were 80 ml of the seed culture solution ofAureobasidium sp. SN-γ96 strain (FERM BP-1431) prepared in accordancewith the procedures of Example 20(a). Culture was then carried out at35° C. and 1000 rpm under an aeration of 2.0 vvm for 15 days.

After the completion of culture, the analysis of the culture solutionindicated that the sucrose was completely consumed, and the erythritoland glycerol contents were 21.6 % (the yield : 23.0 %) and 4.9 % (theyield : 5.3 %), respectively.

EXAMPLE 27

Shown in this example are the results of comparison of the resistance tosugars of the wild Aureobasidium sp. SN-124A strains of the presentinvention and their mutants Aureobasidium sp. SN-G42 strains andAureosbasidium sp. SN-γ96 strains.

Procedures

One hundred (100) ml of each of liquid culture media containing 2.0 % ofyeast extract (manufactured by Difco, Co., Ltd.) and given amounts (22.0to 83.3 %) of glucose were loaded into a conical flask of 500 ml involume, and were sterilized in the conventional manner. Thereafter, theslant-cultured cells of Aureobasidium sp. SN-124A, SN-G42 and SN-γ96strains were inoculated on the respective media for seed culture at 35°C. for 1 to 5 days.

Next, 2 l of each liquid culture media containing given amounts (22.0 to83.3 %) of glucose and 20 % of yeast extract were put in a fermenter of3 l in volume. Added to the medium were 80 ml of each of the aforesaidseed culture solutions having the corresponding substrateconcentrations. Culture was then carried out at a culture temperature of35° C. and at 1000 rpm under an aeration of 2.0 vvm until the glucose ineach medium was completely consumed (for 2 to 14 days).

After the completion of culture, the amount of erythritol contained ineach culture solution was measured by HPLC.

Results

The yields of erythritol per consumed glucose are tabulated as follows:

    ______________________________________                                                    Yields of Erythritol in %                                         Glucose Concentration                                                                       Parent Strains                                                                            Mutants   Mutants                                   in %          (SN-124A)   (SN-G42)  (SN-Υ96)                          ______________________________________                                        22.0          41.5        50.6      48.2                                      33.5          37.0        48.0      45.9                                      39.5          29.7        46.8      47.5                                      45.0          16.2        43.5      45.3                                      53.3          --          40.5      42.5                                      60.2          --          32.4      43.1                                      67.9          --          --        38.6                                      75.5          --          --        32.4                                      83.3                                26.4                                      ______________________________________                                    

EXAMPLE 28

Shown in this example are the results of comparison of the foaming,flocculation and hydrophilic natures of Aureobasidium sp. SN-124A strainand their mutants, Aureobasidium sp. SN-G42 strain and SN-γ96 strain.

Procedures

Five (5) ml of a liquid culture medium containing 1.0 % of yeast extractand 33.5 % of glucose were put into a test tube, and weresteam-sterilized at 120° C. for 15 minutes. After cooling, the mediumwas adjusted to pH 5.5, and was inoculated with the slant-cultured cellsof Aureobasidium sp. SN-124A strain or SN-G42 strain, followed by rotaryshaking culture at 30° C. for 5 days. After the completion of culture,the culture solution was kept stationary for 15 minutes to observe thegeneration of foams and the state of flocculation of the cells (foamingand flocculation natures).

Subsequently, an equal amount of benzene was added under vigorousagitation to the culture solution, and the resulting solution wasthereafter kept stationary for about 30 minutes to observe the transferof the cells into the benzene phase (hydrophilic nature).

Similar observations were also carried out with Aureobasidium sp. SN-γ96strains on a liquid culture containing 1.6 % of yeast extract and 40.0 %of glucose.

Results

When the parent Aureobasidium sp. SN-124A strain was aerobicallycultured on a liquid culture medium, marked foaming occurred. The foamsremained for a longer period of time even after the stopping ofreciprocal shaking. The cells in the culture solution were flocculatedimmediately upon stirring being interrupted, thus giving sediments.

However, even when the mutants, Aureobasidium sp. SN-G42 and SN-γ96strains were cultured under the same conditions as mentioned above, nofoaming was observed. Nor did the flocculation of the cells occur, evenafter the stopping of reciprocal shaking.

Further, when suitable amounts of benzene were added to the respectiveculture solutions of the parent strains and mutants to form double-phasesystems, it was found that the cells of the parent strains were passedinto the benzene phase without leaving no trace in the aqueous phase,but the cells of the mutants were all kept in the aqueous phases withoutpassing into the benzene phases.

What is claimed is:
 1. A biologically pure culture of Aureobasidium sp.SN-124A strain (FERM BP-1429) and artificial mutants thereof, which havethe properties of forming and accumulating erythritol in a culturesolution when aerobically cultured on a liquid culture medium containingan assimilable carbon source and an assimilable nitrogen source, and arecapable of preparing erythritol by fermentation of sugars.
 2. Abiologically pure culture strain of an artificial mutant ofAureobasidium sp. SN-124A strain (BP-1429), which has the properties offorming and accumulating erythritol in a culture solution without anysubstantial occurrence of foams due to fermentation when aerobicallycultured on a liquid medium containing an assimilable carbon source inan amount of at least 40% and an assimilable nitrogen source, producehydrophilic cells, and is capable of preparing erythritol byfermentation of sugars.
 3. The biologically pure culture strain asdefined in claim 1, in which said artificial mutant of Aureobasidium sp.SN-124A strain is Aureobasidium sp. SN-G42 strain
 4. The biological pureculture strain as defined in claim 3, in which said artificial mutant ofAureobasidium sp. SN-124A strain is Aureobasidium sp. SN-γ96 strain(FERM BP-1431).